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Changes in the Acetylome and Succinylome of Bacillus subtilis in Response to Carbon Source.

Kosono S, Tamura M, Suzuki S, Kawamura Y, Yoshida A, Nishiyama M, Yoshida M - PLoS ONE (2015)

Bottom Line: Changes in acetylation and succinylation were observed in proteins involved in central carbon metabolism and in components of the transcription and translation machineries, such as RNA polymerase and the ribosome.Mutations that modulate protein acylation affected B. subtilis growth.Our results suggest that acyl modifications play a role in the physiological adaptations to changes in carbon nutrient availability of B. subtilis.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Center, the University of Tokyo, Bunkyo-ku, Tokyo, Japan; RIKEN Center for Sustainable Resource Science, Wako, Saitama, Japan.

ABSTRACT
Lysine residues can be post-translationally modified by various acyl modifications in bacteria and eukarya. Here, we showed that two major acyl modifications, acetylation and succinylation, were changed in response to the carbon source in the Gram-positive model bacterium Bacillus subtilis. Acetylation was more common when the cells were grown on glucose, glycerol, or pyruvate, whereas succinylation was upregulated when the cells were grown on citrate, reflecting the metabolic states that preferentially produce acetyl-CoA and succinyl-CoA, respectively. To identify and quantify changes in acetylation and succinylation in response to the carbon source, we performed a stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic analysis of cells grown on glucose or citrate. We identified 629 acetylated proteins with 1355 unique acetylation sites and 204 succinylated proteins with 327 unique succinylation sites. Acetylation targeted different metabolic pathways under the two growth conditions: branched-chain amino acid biosynthesis and purine metabolism in glucose and the citrate cycle in citrate. Succinylation preferentially targeted the citrate cycle in citrate. Acetylation and succinylation mostly targeted different lysine residues and showed a preference for different residues surrounding the modification sites, suggesting that the two modifications may depend on different factors such as characteristics of acyl-group donors, molecular environment of the lysine substrate, and/or the modifying enzymes. Changes in acetylation and succinylation were observed in proteins involved in central carbon metabolism and in components of the transcription and translation machineries, such as RNA polymerase and the ribosome. Mutations that modulate protein acylation affected B. subtilis growth. A mutation in acetate kinase (ackA) increased the global acetylation level, suggesting that acetyl phosphate-dependent acetylation is common in B. subtilis, just as it is in Escherichia coli. Our results suggest that acyl modifications play a role in the physiological adaptations to changes in carbon nutrient availability of B. subtilis.

No MeSH data available.


Related in: MedlinePlus

Growth of wild type and mutant strains in glucose or citrate.Fresh colonies grown on minimal glucose plates supplemented with amino acid mixture were inoculated in a modified Spizizen’s minimal medium supplemented with 30 mM glucose (A and C) or 30 mM citrate (B and D). Growth curves were monitored by measuring OD at 660 nm. 168 (WT, black circle), SS110 (ΔacuA, blue triangle), SS38 (ΔacuC ΔsrtN, blue square), SS51 (ΔackA, green triangle), SS52 (Δpta, green square), SS53 (ΔackA Δpta, red triangle), and SS111 (ΔackA Δpta ΔacuA, red square) strains. Panels A and B show the WT and isogenic mutant strains plotted along with the average OD660 values from three growth experiments. Panel C shows SS52 (Δpta), SS53 (ΔackA Δpta), SS110 (ΔacuA), SS111 (ΔackA Δpta ΔacuA), and WT strains along with error bars, as reproduced from panel A. Panel D shows SS110 (ΔacuA) and WT strains along with error bars, as reproduced from panel B.
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pone.0131169.g002: Growth of wild type and mutant strains in glucose or citrate.Fresh colonies grown on minimal glucose plates supplemented with amino acid mixture were inoculated in a modified Spizizen’s minimal medium supplemented with 30 mM glucose (A and C) or 30 mM citrate (B and D). Growth curves were monitored by measuring OD at 660 nm. 168 (WT, black circle), SS110 (ΔacuA, blue triangle), SS38 (ΔacuC ΔsrtN, blue square), SS51 (ΔackA, green triangle), SS52 (Δpta, green square), SS53 (ΔackA Δpta, red triangle), and SS111 (ΔackA Δpta ΔacuA, red square) strains. Panels A and B show the WT and isogenic mutant strains plotted along with the average OD660 values from three growth experiments. Panel C shows SS52 (Δpta), SS53 (ΔackA Δpta), SS110 (ΔacuA), SS111 (ΔackA Δpta ΔacuA), and WT strains along with error bars, as reproduced from panel A. Panel D shows SS110 (ΔacuA) and WT strains along with error bars, as reproduced from panel B.

Mentions: To evaluate the changes in lysine acetylation and succinylation in response to the carbon source, we used a quantitative MS approach based on SILAC. We chose glucose or citrate as the different growth conditions, because we observed different acyl modification patterns with these substrates, as shown in Fig 1. Under these two conditions, the carbon metabolic fluxes are totally different [41]. We chose an OD660 of 0.5 as the growth point at which wild type cell growth was exponential (Fig 2). For glucose-heavy labeling (exp. 1), a B. subtilis lysine auxotroph strain (TM61) was grown in glucose with heavy lysine medium or in citrate with light lysine medium. For citrate-heavy labeling (exp. 2), the strain was grown in glucose with light lysine medium or in citrate with heavy lysine medium. We confirmed with western blot analysis that the lysA mutation in the TM61 strain did not affect the global acetylation and succinylation profiles when compared to those in the wild type strain (S2 Fig). After labeling, lysates from both conditions containing equal amounts of protein were mixed and digested with trypsin. The acetylated or succinylated lysine peptides were then affinity enriched using anti-acetyllysine or anti-succinyllysine antibodies, respectively. We also analyzed the mixed total trypsinized peptides without affinity enrichment to estimate the relative abundance of the proteins under these two conditions. The labeling efficiencies were 99.8% for glucose-heavy labeling and 99.7% for citrate-heavy labeling. The change in modification levels was evaluated by determining the R-value, which was calculated from the ratio of peptide peak areas normalized to the ratio of protein abundance. The data were obtained from duplicate experiments with switched labeling (exp. 1 and exp. 2). The acetylation and succinylation sites identified in each experiment are summarized in S3 Fig.


Changes in the Acetylome and Succinylome of Bacillus subtilis in Response to Carbon Source.

Kosono S, Tamura M, Suzuki S, Kawamura Y, Yoshida A, Nishiyama M, Yoshida M - PLoS ONE (2015)

Growth of wild type and mutant strains in glucose or citrate.Fresh colonies grown on minimal glucose plates supplemented with amino acid mixture were inoculated in a modified Spizizen’s minimal medium supplemented with 30 mM glucose (A and C) or 30 mM citrate (B and D). Growth curves were monitored by measuring OD at 660 nm. 168 (WT, black circle), SS110 (ΔacuA, blue triangle), SS38 (ΔacuC ΔsrtN, blue square), SS51 (ΔackA, green triangle), SS52 (Δpta, green square), SS53 (ΔackA Δpta, red triangle), and SS111 (ΔackA Δpta ΔacuA, red square) strains. Panels A and B show the WT and isogenic mutant strains plotted along with the average OD660 values from three growth experiments. Panel C shows SS52 (Δpta), SS53 (ΔackA Δpta), SS110 (ΔacuA), SS111 (ΔackA Δpta ΔacuA), and WT strains along with error bars, as reproduced from panel A. Panel D shows SS110 (ΔacuA) and WT strains along with error bars, as reproduced from panel B.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4476798&req=5

pone.0131169.g002: Growth of wild type and mutant strains in glucose or citrate.Fresh colonies grown on minimal glucose plates supplemented with amino acid mixture were inoculated in a modified Spizizen’s minimal medium supplemented with 30 mM glucose (A and C) or 30 mM citrate (B and D). Growth curves were monitored by measuring OD at 660 nm. 168 (WT, black circle), SS110 (ΔacuA, blue triangle), SS38 (ΔacuC ΔsrtN, blue square), SS51 (ΔackA, green triangle), SS52 (Δpta, green square), SS53 (ΔackA Δpta, red triangle), and SS111 (ΔackA Δpta ΔacuA, red square) strains. Panels A and B show the WT and isogenic mutant strains plotted along with the average OD660 values from three growth experiments. Panel C shows SS52 (Δpta), SS53 (ΔackA Δpta), SS110 (ΔacuA), SS111 (ΔackA Δpta ΔacuA), and WT strains along with error bars, as reproduced from panel A. Panel D shows SS110 (ΔacuA) and WT strains along with error bars, as reproduced from panel B.
Mentions: To evaluate the changes in lysine acetylation and succinylation in response to the carbon source, we used a quantitative MS approach based on SILAC. We chose glucose or citrate as the different growth conditions, because we observed different acyl modification patterns with these substrates, as shown in Fig 1. Under these two conditions, the carbon metabolic fluxes are totally different [41]. We chose an OD660 of 0.5 as the growth point at which wild type cell growth was exponential (Fig 2). For glucose-heavy labeling (exp. 1), a B. subtilis lysine auxotroph strain (TM61) was grown in glucose with heavy lysine medium or in citrate with light lysine medium. For citrate-heavy labeling (exp. 2), the strain was grown in glucose with light lysine medium or in citrate with heavy lysine medium. We confirmed with western blot analysis that the lysA mutation in the TM61 strain did not affect the global acetylation and succinylation profiles when compared to those in the wild type strain (S2 Fig). After labeling, lysates from both conditions containing equal amounts of protein were mixed and digested with trypsin. The acetylated or succinylated lysine peptides were then affinity enriched using anti-acetyllysine or anti-succinyllysine antibodies, respectively. We also analyzed the mixed total trypsinized peptides without affinity enrichment to estimate the relative abundance of the proteins under these two conditions. The labeling efficiencies were 99.8% for glucose-heavy labeling and 99.7% for citrate-heavy labeling. The change in modification levels was evaluated by determining the R-value, which was calculated from the ratio of peptide peak areas normalized to the ratio of protein abundance. The data were obtained from duplicate experiments with switched labeling (exp. 1 and exp. 2). The acetylation and succinylation sites identified in each experiment are summarized in S3 Fig.

Bottom Line: Changes in acetylation and succinylation were observed in proteins involved in central carbon metabolism and in components of the transcription and translation machineries, such as RNA polymerase and the ribosome.Mutations that modulate protein acylation affected B. subtilis growth.Our results suggest that acyl modifications play a role in the physiological adaptations to changes in carbon nutrient availability of B. subtilis.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Center, the University of Tokyo, Bunkyo-ku, Tokyo, Japan; RIKEN Center for Sustainable Resource Science, Wako, Saitama, Japan.

ABSTRACT
Lysine residues can be post-translationally modified by various acyl modifications in bacteria and eukarya. Here, we showed that two major acyl modifications, acetylation and succinylation, were changed in response to the carbon source in the Gram-positive model bacterium Bacillus subtilis. Acetylation was more common when the cells were grown on glucose, glycerol, or pyruvate, whereas succinylation was upregulated when the cells were grown on citrate, reflecting the metabolic states that preferentially produce acetyl-CoA and succinyl-CoA, respectively. To identify and quantify changes in acetylation and succinylation in response to the carbon source, we performed a stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic analysis of cells grown on glucose or citrate. We identified 629 acetylated proteins with 1355 unique acetylation sites and 204 succinylated proteins with 327 unique succinylation sites. Acetylation targeted different metabolic pathways under the two growth conditions: branched-chain amino acid biosynthesis and purine metabolism in glucose and the citrate cycle in citrate. Succinylation preferentially targeted the citrate cycle in citrate. Acetylation and succinylation mostly targeted different lysine residues and showed a preference for different residues surrounding the modification sites, suggesting that the two modifications may depend on different factors such as characteristics of acyl-group donors, molecular environment of the lysine substrate, and/or the modifying enzymes. Changes in acetylation and succinylation were observed in proteins involved in central carbon metabolism and in components of the transcription and translation machineries, such as RNA polymerase and the ribosome. Mutations that modulate protein acylation affected B. subtilis growth. A mutation in acetate kinase (ackA) increased the global acetylation level, suggesting that acetyl phosphate-dependent acetylation is common in B. subtilis, just as it is in Escherichia coli. Our results suggest that acyl modifications play a role in the physiological adaptations to changes in carbon nutrient availability of B. subtilis.

No MeSH data available.


Related in: MedlinePlus